amoeba lecture
TRANSCRIPT
Entamoeba histolytica
Wrap up nematodes with Dracunculus
Intro into protozoa
Entamoeba, cell biology, disease, and treatment
Why do some amoebae cause disease and others not?
From Exercitationes de Vena Mediensis et de Vermiculis Capilaribus Infantium by G.H. Velschius, published in Augsburg, Germany 1674
dracunculiasis
Guinea worm infection caused by Dracunculus mediensis
3 million cases in 1992 prior to start of eradication program
Now down to 10 thousands, most of them in the Sudan
dracunculiasis
dracunculiasis
Formation of a blister which can burn or itch strongly
The blister bursts and gives rise to an ulcer with the female protruding (triggered by cooling through water contact)
Frequent secondary bacterial infections
dracunculiasis
Embryos are squeezed out in response to cold water
dracunculiasis
dracunculiasis
Drancunculiasis is a preventable disease:
No reservoir Prevent contact of infected
people with water sources Filter water to remove
intermediate host Relatively low cost educational
campaign Go to the web site of the
Carter Center to learn more about this highly successful control/eradication campaign
protozoa
Primary unicellular eukaryotes, often also called protists
Many important human and veterinary pathogens
It is important to understand that protozoa is mostly a historic grouping and not a cohesive biological group that contains closely related organisms
A very diverse group with a vast variety of morphological and biochemical adaptations to almost any ecological niche
the tree of life(Ernst Haeckel, 1874)
protozoa
reptiles
molluscs
man
crustaceans
whales
fish
worms
carnivoresungulates The tree of life (who is
related and how did they evolve) was initially based on morphological characteristics
“Complex” organisms were viewed as derived and highly evolved “simple” organisms as primitive
This scheme puts protozoa as a cohesive group to the bottom of the tree
The three kingdoms of life(Mitch Sogin’s 16s RNA tree)
While the protozoan phyla as evolutionary groups are well defined how they related to each other is not well understood
The most recent models of eukaryotic evolution propose an early split giving rise to animals, fungi and amoeba on one side and plants and most of the other protozoans on the other
Who came first on this tree, who is the most primitive and what the relationships among the phyla are is not well defined
amoebae the most primitive eukaryotes?
Classical taxonomy puts amoebae at the very base of the eukaryotic tree as they lack many derived morphological characters
No special organelles for locomotion (cilia, flagella)
No mitochondrion and no typical mitochondrial enzymes
A fermentative “bacteria-like” anaerobic metabolism
It was assumed that amoeba represent the stage of early eukaryotes before the endosymbiosis event that let to the mitochondrion
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Is the absence of mitochondria a primary of secondary trait?
The genomes of most important protozoan parasites are now fully sequenced
This provides the opportunity to hunt for ‘molecular fossils’
Most proteins that do their job in the mitochondrion are actually encoded in the nucleus and are imported from the cytoplasm
Genome searches in Entamoeba identified several genes for proteins targeted to the mitochondrion in other organisms
Antibodies generated against these proteins identified a small highly reduced mitochondrion - the mitosom
This suggest amoebae had full-fledged mitochondria in the past but reduced them when they moved to an anaerobic environment
For the moment we don’t know a eukaryote featuring a primary lack of mitochondria
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what is amoeboid about amoebae?
Amoeboid movement
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Acanthamoebahttp://cmgm.stanford.edu/theriot/movies.htm#Hits
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what is amoeboid about amoebae?
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Uroid
Pseudopodium
Hyaline ectoplasm
Endoplasm (sol)
While the endoplasm (sol) is ‘liquid’ andfilled with organelles the ectoplasms appearsmore solid (gel) and clear.
(gel)
Amoeboid movement is not limited to amoeba
Neutrophil chasing a bacterium
What could be the engine and gears powering this movement?
Muscle: actin provides structure
but myosin is the motor
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Ameboid movement is driven by actin
Amoeboid movement depends on the actin cytoskelleton
Earlier models were based on cortical actin/myosin squeezing the cytoplasm to the leading edge (toothpaste tube model), this was thought to be accompanied by cytoplasmic gel/sol transformations
More recent data strongly support actin polymerization as the force generating step (at least for the best understood part of protrusion)
Actin dynamics in amoeboid movement are complex and not easily dissected -- can just polymerizing actin really drive motility?
Listeria as a model to demonstrate and study actin polymerization motility
http://cmgm.stanford.edu/theriot/movies.htm#Hits
Listeria in host cell (150x)
Listeria in Xenopus extract (right panel
Phase contrast, left panel actin-GFP fluorescence)
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The actin polymerization model is based on cell free reconstitution of the movement of intracellular bacteria
These studies allowed to identify the factors involved in the initiation of actin filament polymerization
Entamoeba histolytica
Fedor Alexandrewitch Lösch describes amoebae associated with severe dysentery in a patient in 1873
He transferred amoebae to a dog by rectal injection, which became ill and showed ulceration of colon
Patient who died from infection showed similar ulcers upon autopsy
trophozoites and cysts
trophozoites and cysts
multiple well defined pseudopodia often extended eruptively
Differentiation into endo- and ectoplasm
Spherical nucleus (4-7 m) with small central nucleolus and characteristic radial spokes
trophozoites and cysts
Trophozoites 20-40 m diameter
Ribosomes arranged in helical patterns
Tissue forms often contain phagocytized red blood cell
trophozoites and cysts
Trophozoites encyst and cysts mature as they travel through the colon
Only mature cysts are infective
trophozoites and cysts
Chromidial bodies and bars are semicrystalline arrays of riobosomes
Round (10- 16 m), 4 nuclei
150 nm cyst wall with fibrillar structure
Entamoeba cysts (light microscopy)
E. coli E. histolytica
Human infection
Major sources for human infection are contamination of drinking water and vegetables (fertilization with material containing or contaminated with human feces)
Patients without any symptoms might nevertheless shed large amounts of cysts
If kept cool and moist (water or soil) cysts can stay infectious for up to a month
Cysts are fairly resistant to chlorination of drinking water (10 mg/l versus 0.1 - 1.0 mg/l for enteric bacteria)
Humans harbor numerous amebae (most are nonpathogenic)
Humans harbor numerous amebae (most are nonpathogenic)
Colitis is the most common form of disease associated with amoebae
Gradual onset of abdominal pain, watery stools containing mucus and blood
Some patients have only intermittent diarrhea alternating with constipation
Fever is uncommon Formation of ulcers
Colitis is the most common form of disease associated with amoebae
Amoeba invade mucosa and erode through laminia propria causing characterisitic flask shaped ulcers contained by muscularis
Ulceration can lead to secondary infection and extraintestinal lesions
Extraintestinal amebiasis
Amebic liver abscess
Most common form of extraintestinal amebiasis
Fast growing abscess filled with debris, amoebae are found only at borders
Lead symptoms are are right upper quadrant pain and fever
Acute as well as chronic illness, with gradual or sudden onset
Amebic liver abscess
30-50% of patients with liver abscess show also pneumonic involvement
Rupture is again a major thread, especially rupture into the pericardium
Draining abscesses is today only performed in extreme cases when rupture is feared
Responds well to chemotherapy
Metronidazole is the drug of choice for extra-intestinal amebiasis
Several drugs are available to clear symptomatic and asymptomatic enteric (luminal) infection (e.g. dichloroacetamides which have unknown mode of action)
Metronidazole (Flagyl) is the drug of choice for invasive amoebiasis (and should be combined with a lumen acting drug as it is not fully effective on luminal stages)
Metronidazole is a prodrug which is activated by an enzyme involved in the microaerobic fermentation metabolism of E. histolytica
Amoebae use fermentation
“La fermentation est la vie sans l’air” (Louis Pasteur)
Entamoeba lacks a functional Krebs cycle and oxidative phosphorylation
Final endproducts of E. histolytica fermentation are CO2, acetate, ethanol and alanine
Metronidazole is activated by PFOR
acetate
CO2
ADPATP
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Entamoeba uses a pyruvate ferredoxin oxidoreductase (PFOR) to break down pyruvate
This process depends on the absence (or low level) of oxygen
This enzyme system is limited to anaerobic bacteria and some protozoa and humans lack this enzyme
PFOR and ferredoxin can transfer an electron to metronidazole producing a highly toxic nitroradical
Drugs which are not toxic but have to be activated into a toxic compound are called prodrugs (look at this as a suicide substrate)
Epidemiology of Entamoeba
480,000,000 people harbor Entamoeba36,000,000 develop clinical symptoms40,000 - 100,000 deaths per year
(Walsh, 1986, Rev. Infect. Dis., based on 1981 data, no significant change since then)
Less than 10% of the people infected show disease. Several hypotheses have been put forward to explain this differential pathogenesis.
Commensal hypothesis
E. histolytica usually is a benign gut commensal as many other amoebae (minuta form)
A certain stimulus (gut flora, diet, host immune status …) transforms the organism into a pathogen (magna form, Kuenen, 1913)
This has been the accepted view for most of the 20th century
Two species hypothesis There are two morphologically
indistinguishable species: E. histolytica and E. dispar. Only one of them (hystolytica) causes disease while the other is benign (Brumpt, 1928)
This theory was discounted and ridiculed
Recent molecular data have revived the two species hypothesis
We now know that most people are infected with the apathogenic E. dispar
Emile Brumpt 1877-1951
Genetic evidence for two species
Species specific isoenzyme patternsMultiple antibodies specific for either the
pathogenic or apathogenic speciesNumerous genes sequenced which show clear
differencesRepetitive DNA elements are differentGenomic organization of conserved gene loci
like actin is differentRibosomal RNA (2.2% difference)
Pathogenic amoeba show contact dependent killing
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Pathogenic amoeba show contact dependent killing
Entamoeba pathogenesis factors
What are pathogen proteins (and genes) that are required to cause disease?
Several candidates have been studied for their involvement in contact dependent cell killing by amoeba:
The surface lectins: These are proteins that allow the amoeba to bind to sugars on the surface of cells and establish tight contact
Proteases: several protein degrading enzymes have been linked to tissue penetration and liver abscess formation
Amoebapores: protein toxins that perforate target cells
Amoebapores one of the candidate pathogenicity factors
Family of small (77 AA) proteins contained in secretory granules
Similar in structure and function to NK lysins
Used to kill bacteria and host cells
Amoebapores insert into target membranes and form ion channels
Amoeba mutants which make less amoebapores cause less disease in animal model studies